Nature Immunology
4, 857 - 865 (2003)
Published online: 10 August 2003; | doi:10.1038/ni963
Defective development and function of Bcl10-deficient follicular, marginal zone and B1 B cellsLiquan Xue1, Stephan W Morris1, 2, 4, Carlos Orihuela3, Elaine Tuomanen3, Xiaoli Cui1, Renren Wen5
& Demin Wang5, 6, 71 Department of Pathology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA. 2 Department of Hematology/Oncology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA. 3 Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA. 4 Department of Pediatrics, University of Tennessee, College of Medicine, Memphis, Tennessee 38163, USA. 5 Blood Research Institute, The Blood Center of Southeastern Wisconsin, Milwaukee, Wisconsin 53226, USA. 6 Model Animal Research Center, Nanjing University, Nanjing, China. 7 Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Correspondence should be addressed to Stephan W Morris steve.morris@stjude.org or Demin Wang dwang@bcsew.eduBcl10 is an intracellular protein essential for nuclear factor (NF)- B activation after lymphocyte antigen receptor stimulation. Using knockout mice, we show that absence of Bcl10 impeded conversion from transitional type 2 to mature follicular B cells and caused substantial decreases in marginal zone and B1 B cells. Bcl10-deficient B cells showed no excessive apoptosis. However, both Bcl10-deficient follicular and marginal zone B cells failed to proliferate normally, although Bcl10-deficient marginal zone B cells uniquely failed to activate NF-B efficiently after stimulation with lipopolysaccharide. Bcl10-deficient marginal zone B cells did not capture antigens, and Bcl10-deficient (Bcl10-/-) mice failed to initiate humoral responses, leading to an inability to clear blood-borne bacteria. Thus, Bcl10 is essential for the development of all mature B cell subsets.After successful rearrangement of both their heavy- and light-chain immunoglobulin genes, B cell precursors express a surface immunoglobulin receptor (IgM) and are defined as immature B cells. These immature B cells then become subject to further positive and negative selection, and some cells eventually give rise to mature B cells1,
2. Based on cell surface markers, anatomical localization and function, mature B cells are classified into at least three subsets3. The recirculating B2 B cells, also called follicular B cells, localize to the B lymphoid follicles of the spleen and lymph node4. The mostly nonrecirculating marginal zone B cells reside mainly around the periphery of the splenic lymphoid nodules3,
5. The nonrecirculating B1 B cells are enriched in the peritoneal and pleural cavities6. Marginal zone and B1 B cells contribute substantially to the initial rapid T cell−independent IgM antibody response and form a first line of defense against antigens7,
8. In contrast, follicular B cells participate later in the T cell−dependent antibody responses7.
The molecular mechanisms by which B cell precursors are instructed to differentiate into different subsets of mature B cells are not well understood. Although T cells are not required, signals from the B cell receptor are known to be involved in the development of follicular, marginal zone and B1 B cells3,
7,
9. Many genes seem to be differentially involved in the development of the three mature B cell subsets3,
9.
Bcl10 is an intracellular signaling protein originally identified because of its involvement in the t(1; 14) (p22; q32) chromosomal translocation that occurs specifically in the mucosa-associated lymphoid tissue (MALT) lymphomas10,
11. Bcl10 contains a caspase recruitment domain (CARD) that participates in the regulation of cellular apoptosis and activation of NF- B10,
11. The exact mechanisms by which Bcl10 activates NF-B are still being clarified, but they seem to involve at least in part physical interactions with the CARD-containing protein TRAF2 (refs. 12,13) and the membrane-associated guanylate kinase family members CARD9 (ref. 14), CARD10 (ref. 15; also known as Bimp1; ref. 16), CARD11 (refs. 17,18; also known as Carma1; refs. 19,20) and CARD14 (ref. 18). These interactions with membrane-associated guanylate kinase family members are thought to organize Bcl10 and other signaling proteins in NF-B-activating pathways emerging from certain cell surface receptors; for example, CARD11 (Carma1) physically associates with the T cell receptor (TCR) and Bcl10 after TCR stimulation, and a CARD11 (Carma1) mutant defective for Bcl10 binding has a dominant-negative effect on TCR-induced NF-B activation19. Consistent with these data supporting the idea of involvement of Bcl10 in cell surface−proximal signaling aspects of NF-B activation, other studies have suggested the protein acts upstream of IB21,
22. In addition to those factors that positively enhance the effects of Bcl10 on NF-B function, negative regulators have been identified that alter NF-B activation by Bcl10 (refs. 12,21,22). The interaction of Bcl10 with several signaling partners in the regulation of NF-B activity indicates that it may be essential in multiple physiological functions.
Studies using Bcl10-deficient mice have shown that Bcl10 is essential for neural tube development and lymphocyte activation23. Bcl10-deficient lymphocytes showed a failure to proliferate in response to B or T cell antigen receptor stimulation, an effect due at least in part to the complete absence of NF-B activation that normally occurs after initiation of signaling from these receptors. Bcl10 deficiency led to a reduction of CD4CD8 double-positive thymocytes and impaired function of both T and B cells, resulting in severe immunodeficiency. Nonetheless, those studies did not detect any B cell developmental defects in the Bcl10-deficient mice23. Here, we have independently generated Bcl10-deficient mice and show that Bcl10 is essential not only for the function but also for the maturation of B cells. Bcl10 deficiency resulted in a considerable reduction in follicular, marginal zone and B1 B cell numbers. In addition, Bcl10 deficiency rendered marginal zone B cells unable to fully proliferate or activate NF-B in response to stimulation by lipopolysaccharide (LPS) and unable to capture blood-borne antigens. Furthermore, Bcl10-deficient mice failed to respond to either T cell−dependent or T cell−independent antigens, to clear bacteria from their bloodstream or to survive infection with the bacterial pathogen Streptococcus pneumoniae. Thus, Bcl10 is important in both the normal maturation and function of follicular, marginal zone and B1 B cells.
Results
Generation of Bcl10-deficient mice
We disrupted Bcl10 in mouse embryonic stem (ES) cells by replacing the third exon, which encodes almost the entirety of the single Bcl10 CARD domain, with a neomycin resistance expression cassette (neo; Fig. 1a). We injected ES cell clones containing a correctly targeted Bcl10 allele into C57BL/6 blastocysts. We obtained germline transmission from two independent ES cell clones, and the mouse lines had identical phenotypes. Heterozygous mice were phenotypically normal and were bred to obtain mice homozygous for the mutated allele. The absence of functional Bcl10 resulted in the death of 30% of homozygous-null embryos between day 18.5 (E18.5) and birth, because of a neural tube closure defect in the hindbrain with exencephaly (data not shown), which was identical to that described before23. Homozygous-null mice that were born viable showed no gross anatomical defects and were fertile. We assessed homozygous-null mice for Bcl10 protein by immunoblot analysis of whole-cell lysates derived from bone marrow, thymus, lymph nodes and spleen using a monoclonal antibody directed to the C terminus of Bcl10. The homozygous-null mutant mice had no detectable Bcl10 protein expression (Fig. 1b).
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Bcl10 deficiency impairs follicular B cell maturation
Previous studies of Bcl10-deficient mice failed to identify any defects of B cell development23. We examined in greater detail the development of pro-B cells, pre-B cells, immature B cells and mature B cells in bone marrow derived from Bcl10-deficient mice. The population of total B cells (B220+) was decreased in bone marrow derived from Bcl10-deficient mice relative to that from wild-type mice (wild-type, 57.8  12.2%, versus Bcl10-/-, 39.3 8.7%; n = 6; P < 0.01; Fig. 2a). Although the reduction in total B cells was relatively modest, we found it consistently in the Bcl10-/- mice examined. The population of B220+IgM- cells, which contains both pro-B and pre-B lymphocytes, was similar in bone marrow derived from Bcl10-deficient and wild-type mice (Fig. 2b). In gated IgM- lymphocytes, the populations of pro-B cells (B220+CD43+IgM-) and pre-B cells (B220+CD43-IgM-) were also similar in Bcl10-deficient and wild-type mice (data not shown). The normal early B cell development in Bcl10-deficient mice agrees with our finding that bone marrow from Bcl10-deficient mice, relative to that of wild-type mice, has normal numbers of interleukin 7 (IL-7)-responsive B cell colonies (data not shown). Furthermore, the populations of immature B lymphocytes (IgM+B220int) were indistinguishable between Bcl10-deficient and wild-type mice (Fig. 2b). In contrast, the population of recirculating mature B cells (IgM+B220hi) was significantly decreased in the bone marrow of Bcl10-deficient mice relative to that in the bone marrow of wild-type mice (wild-type, 8.9 2.7%, versus Bcl10-/-, 2.4 0.7%; n = 5; P < 0.01; Fig. 2b). The reduction in recirculating B cells in the bone marrow of Bcl10-deficient mice indicates that the lack of normal Bcl10 function might impair B cell maturation.
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New B cell precursors emigrate from the bone marrow to mature within the spleen2,
24. We therefore next examined the effect of Bcl10 deficiency on splenic B cell differentiation. The total number of splenocytes tended to be slightly lower in mice lacking Bcl10 than in wild-type mice, although the difference did not reach statistical significance (wild-type, 113.1  106 37.0 106 cells per spleen, versus Bcl10-/-, 101.9 106 30.7 106 cells per spleen; n = 15; P = 0.36). In contrast, the population of B220+ B cells was consistently and significantly decreased in spleens from Bcl10-deficient mice relative to that of wild-type mice (wild-type, 65.8 10.0%, versus Bcl10-/-, 46.4 5.4%; n = 4; P < 0.01; Fig. 2c). This decrease was consistent with the percentage reduction in total B cells we found in bone marrow from Bcl10-/- mice. To elucidate the deficit in B220+ B cells, we next examined the different subsets of immature and mature B cells present in spleens of Bcl10-/- mice. We stained splenocytes from Bcl10-deficient and wild-type mice with antibody to IgM (anti-IgM), anti-CD21 and anti-CD23, then separated them into CD23+ and CD23- populations. Among the populations of cells gated on CD23+, Bcl10-deficient mice had a significantly decreased population of follicular B cells (CD23+CD21intIgMlo) compared with that of wild-type mice (wild-type, 26.3 2.0%, versus Bcl10-/-, 11.1 3.6%; n = 4, P < 0.01), whereas the population of transitional type 2 B cells (CD23+CD21hiIgMhi) was slightly increased relative to that of wild-type mice (wild-type, 7.1 3.2%, versus Bcl10-/-, 11.6 1.2%; n = 4; P < 0.05; Fig. 2d). In cells gated on CD23-, transitional type 1 B cell (CD23-CD21loIgMhi) percentages in Bcl10-deficient mice were similar to those of wild-type mice (wild-type, 2.7 1.1%, versus Bcl10-/-, 1.8 0.6%; n = 4; Fig. 3a). These data show that deficiency in Bcl10 impairs the transition from transitional type 2 to mature follicular B cells, resulting in a decrease in mature follicular B cells.
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Bcl10 absence impairs marginal zone and B1 B cell development
In addition to mature follicular B cells, the other mature B lymphocytes that reside mainly in the spleen are the marginal zone B cells3,
5. Marginal zone B cells, like transitional type 2 B cells, are IgMhi and CD21hi, but do not express CD23 or IgD2,
25. To examine the effect of Bcl10 deficiency on the development of marginal zone B cells, we stained splenocytes from Bcl10-deficient and wild-type mice with anti-IgM, anti-CD21 and anti-CD23, then separated them into CD23+ and CD23- populations. In the cell population gated on CD23-, Bcl10-deficient mice had a considerably decreased population of marginal zone B cells (CD23-CD21hiIgMhi) relative to that of wild-type mice (wild-type, 3.1 1.2%, versus Bcl10-/-, 0.7 0.3%; n = 4; P < 0.01; Fig. 3a). In addition, the marginal zone B cell population could be recognized as CD19+CD21hiCD23lo cells based on expression of the CD19, CD21 and CD23 markers. In spleen cells gated on CD19+, the population of marginal zone B cells (CD21hiCD23lo) was substantially decreased in Bcl10-deficient mice (wild-type, 8.0 2.5%, versus Bcl10-/-, 2.4 1.1%; n = 12; P < 0.01; Fig. 3b). These flow cytometric data show that Bcl10 deficiency impairs the development of marginal zone B cells.
We further confirmed the considerable reduction of marginal zone B cells by immunofluorescence staining. We stained frozen spleen sections from Bcl10-deficient and wild-type mice with tetramethyl-rhodamine isothiocyanate (TRITC)-conjugated goat anti-mouse IgM and FITC-conjugated rat anti-mouse MOMA-1, a marker specific for metallophilic macrophages. The ring of metallophilic macrophages permits visualization of the border between the follicular and marginal zones. In agreement with our flow cytometric results, the width of marginal zone B cell area was barely detectable in spleens derived from Bcl10-deficient mice (Fig. 4). These results confirmed the physical disappearance of marginal zone B cells in Bcl10-deficient mice despite a normal follicular architecture.
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The self-renewing mature B1 B cells reside mainly in the peritoneal and pleural cavities. To examine the effect of Bcl10 deficiency on the development of B1 cells, we stained peritoneal lymphocytes from Bcl10-deficient or wild-type mice with anti-CD5 and anti-IgM. We found a substantial decrease in B1 B cells in Bcl10-deficient mice (wild-type, 25.6 10.5%, versus Bcl10-/-, 2.5 1.1%; n = 5; P < 0.01; Fig. 3c). Therefore, Bcl10 deficiency impairs the development of B1 B cells.
B cell-intrinsic defect in Bcl10-/- marginal zone lymphocytes
The defect of marginal zone B cell development in Bcl10-deficient mice could be the result of an intrinsic abnormality of the B cells or of an abnormality of the splenic microenvironment. To distinguish these two possibilities, we transplanted equal numbers of bone marrow cells from wild-type or Bcl10-deficient mice into sublethally irradiated mice deficient in recombination activating gene 1 (RAG-1). We analyzed the development of marginal zone B cells 8 weeks after the transplantation. RAG-1-deficient recipients that received wild-type bone marrow cells had normal marginal zone B cell development (Fig. 5). In contrast, RAG-1-deficient mice that received Bcl10-deficient bone marrow cells had substantially reduced numbers of marginal zone B lymphocytes (CD21hiCD23lo), similar to the defect found in Bcl10-deficient mice. Moreover, bone marrow cells derived from wild-type mice were able to reconstitute the marginal zone B cell population in Bcl10-deficient recipients (Fig. 5). These data demonstrated that the splenic microenvironment in Bcl10-deficient mice was able to support normal marginal zone B cell development and that the marginal zone B cell defect in Bcl10-deficient mice was B cell autonomous.
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Low proliferation of Bcl10-/- follicular and marginal zone B cells
To determine whether the defective development of follicular and marginal zone B cells in Bcl10-deficient mice was because of a failure of their production or survival, we first examined the rate of apoptosis in naive mutant follicular and marginal zone B cells. We stained splenocytes from wild-type or Bcl10-deficient mice with anti-CD21 and anti-CD23, and then analyzed them for apoptosis by TUNEL assay. Naive Bcl10-deficient follicular (CD21+CD23+) and marginal zone (CD21hiCD23lo) B cells had similarly low rates of apoptosis relative to those of the corresponding wild-type B cell subpopulations (Fig. 6a). Both wild-type and Bcl10-deficient follicular and marginal zone B cells showed similar degrees of apoptosis even after stimulation with anti-IgM or LPS (data not shown). These data indicate that Bcl10 deficiency has no substantial effect on the survival rates of either naive follicular or marginal zone B cells.
| | Figure 6. Normal apoptosis in naive Bcl10-deficient follicular and marginal zone B cells, and inability of Bcl10-deficient marginal zone B cells to proliferate or activate NF-B in response to LPS. | | |  | (a) Normal apoptosis in naive Bcl10-deficient follicular (FO) and marginal zone (MZ) B cells. Splenocytes from wild-type (Bcl10+/+) and Bcl10-deficient (Bcl10-/-) mice were stained with CD21/CD23, then the degree of TUNEL labeling in follicular (CD21+CD23+) and marginal zone (CD21hiCD23lo) B cells was determined by FACS analysis. Negative controls were B cells treated as described above but without terminal transferase. TUNEL-positive cells were defined as those showing fluorescence greater than negative controls. Percentages indicate TUNEL-positive cells in the gated B cell subpopulations. Data are representative of three independent analyses. (b) Reduced BrdU incorporation in Bcl10-deficient follicular (FO) and marginal zone (MZ) B cells in vivo. Wild-type (Bcl10+/+) and Bcl10-deficient (Bcl10-/-) mice were injected with BrdU every 12 h for 4 d (4 days); mice that were not injected with BrdU served as the baseline control (0 day). Splenocytes from the mice were stained with purified rat anti-mouse CD21 followed by PE-conjugated anti-rat Ig and biotin-conjugated anti-CD23, and finally by Cy-chrome-labeled streptavidin. The cells were then permeabilized, fixed and stained with FITC-conjugated anti-BrdU. The degree of BrdU positivity in the gated B cell subpopulations, follicular (CD21+CD23+) and marginal zone (CD21hiCD23lo), was analyzed by FACS. Percentages indicate BrdU-positive cells in the gated B cell subpopulations. Data are representative of four mice per genotype. (c) Failure of Bcl10-deficient marginal zone (MZ), but not follicular (FO), B cells to proliferate in response to LPS. Based on CD21 and CD23 expression, marginal zone and follicular B cells were purified by FACS from the splenocytes of wild-type (Bcl10+/+) or Bcl10-deficient (Bcl10-/-) mice, then stimulated with LPS. Proliferative responses were determined by [3H]thymidine incorporation. Data are representative of two independent experiments. (d) Defective NF-B activation by LPS in Bcl10-deficient marginal zone (MZ), but not follicular (FO), B cells. Follicular and marginal zone B cells were purified from individual wild-type (+/+) or Bcl10-deficient (-/-) mice by FACS sorting, followed by stimulation with medium alone (Med) or LPS. Nuclear extracts (1  g/sample) were used for NF-B gel-mobility-shift assays. NF-B gel-shift results obtained from two sets of Bcl10-deficient and wild-type marginal zone B cells after LPS stimulation are shown to emphasize the reproducibility of the diminished response found in the cells lacking Bcl10. Data are representative of three independent experiments.
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To directly assess the production of Bcl10-deficient follicular and marginal zone B cells, we used an in vivo bromodeoxyuridine (BrdU) labeling assay, determining the BrdU-labeling rate of the B cell subpopulations by fluorescence-activated cell sorting (FACS) analysis. Bcl10-deficient follicular and marginal zone B cells had much lower BrdU-labeling rates than did the corresponding wild-type B cell subpopulations (Fig. 6b). Bcl10-deficient marginal zone B cells were especially hypoproliferative relative to wild-type marginal zone B cells, with only 0.9% of the Bcl10-/- cells incorporating BrdU compared with 5.1% of the control cells 4 d after BrdU exposure in a representative experiment (P = 0.003; Fig. 6b). However, Bcl10-deficient follicular B cells showed abnormally low proliferation as well; a typical experiment showed 2.6% of these cells, compared with 7.2% of wild-type follicular B cells, incorporated the BrdU label (P = 0.03). These data indicate that a normal cellular death rate but an abnormally low generation rate exist in Bcl10-deficient follicular and marginal zone B cells in vivo. Bcl10-deficient mice had too few B1 B cells for reliable analysis by TUNEL assay for their apoptotic responses or by in vivo BrdU labeling to assess their baseline proliferation, or for other analyses.
LPS-induced marginal zone B cell proliferation requires Bcl10
Next, we examined the proliferation of Bcl10-deficient B cells in response to LPS. Functionally, marginal zone B cells proliferate strongly in response to LPS, a stimulus that activates B cells through Toll-like receptors26, whereas follicular B cells respond weakly to LPS stimulation3. Previous work showed that Bcl10-deficient, unfractionated, total splenic B cells failed to proliferate in response to stimulation with anti-IgM, but proliferated normally after stimulation with LPS23. However, this LPS response of Bcl10-deficient splenic B cells could be due to normal LPS responses of the follicular B cells, which comprise the main portion of the splenic B cells, but not of the marginal zone B cells, which normally represent only a small percentage of the total splenic B cells. To test this possibility, we sorted Bcl10-deficient marginal zone and follicular B cells by FACS based on CD21 and CD23 expression and examined their abilities to respond to LPS. The proliferation of Bcl10-deficient marginal zone B lymphocytes in response to stimulation with LPS was much less than that of wild-type marginal zone B cells (Fig. 6c). In contrast, the LPS-induced proliferation of Bcl10-deficient follicular B cells was similar to that of wild-type follicular B cells (Fig. 6c). These results demonstrate that Bcl10 deficiency uniquely affects the ability of marginal zone, but not follicular, B cells to respond to LPS.
Impaired NF-B response to LPS in Bcl10-/- marginal zone cells
The NF-B family of transcription factors is important in both the development and function of marginal zone and follicular B cells27. To elucidate the molecular basis for the impairment of LPS signaling in Bcl10-deficient marginal zone B cells, we examined NF-B activation by LPS in wild-type and Bcl10-deficient marginal zone B cells. LPS-induced NF-B activation was substantially reduced in Bcl-10-deficient marginal zone B cells compared with that of wild-type marginal zone B cells (Fig. 6d). In contrast, NF-B activation in response to LPS was similar in purified wild-type and Bcl10-deficient follicular B cells (Fig. 6d). However, IgM cross-linking activated NF-B in wild-type, but not in Bcl10-deficient, follicular B cells (data not shown), consistent with previous findings23. These results demonstrate that Bcl10 is required for normal NF-B activation by LPS-induced signaling specifically in marginal zone B cells.
Bcl10-deficient marginal zone B cells fail to capture antigens
Marginal zone B cells are involved mainly in the early and immediate response to antigenic challenges independent of T cells, and form the first line of defense against blood-borne antigens7. One ability required for marginal zone B cells to accomplish their functions is the efficient capture of particulate antigens in a nonspecific way. To examine the ability of Bcl10-deficient marginal zone B cells to capture antigens, we injected Bcl10-deficient mice intravenously with 2,4,6-trinitrophenol (TNP)-Ficoll, a T cell−independent antigen. Then, 30 min after the injection, we isolated splenocytes and stained them with anti-CD19, anti-CD21, anti-CD23 and anti-TNP. We evaluated antigen capture by examining the presence of TNP-Ficoll on the gated marginal zone and non-marginal zone B cell populations. Bcl10-deficient marginal zone B cells were unable to capture TNP-Ficoll (Fig. 7a). These data demonstrate that Bcl10-deficient marginal zone B cells are functionally disabled.
| | Figure 7. Inability of Bcl10-deficient marginal zone B cells to capture antigens, complete absence of humoral immune responses to T cell−independent− and T cell−dependent−specific antigens, and high susceptibility of Bcl10-deficient mice to bacterial infection. | | |  | (a) Inability of Bcl10-deficient marginal zone B cells to capture antigens. Wild-type (Bcl10+/+) and Bcl10-deficient (Bcl10-/-) mice were injected intravenously without (not immunized) or with TNP-Ficoll. Then, 30 min after injection, splenocytes were prepared and stained with anti-CD19, anti-CD21, anti-CD23 and anti-TNP and were analyzed by FACS. In cells gated on CD19+, marginal zone B cells (CD21hiCD23lo) and non-marginal zone B cells were separated as described for Figure 3b. Histograms show the anti-TNP staining of the gated marginal zone B cells (filled) and non-marginal zone B cells (open). Data are representative of six mice per genotype. (b) Complete absence of the humoral immune response to T cell−independent antigens in Bcl10-deficient mice. Wild-type (Bcl10+/+) and Bcl10-deficient (Bcl10-/-) mice were immunized intraperitoneally with the T cell−independent antigen TNP-Ficoll. Then, 7 d after immunization, sera were collected and TNP-specific immunoglobulin isotypes were determined by ELISA. Data are from three mice per genotype. (c) Complete absence of the humoral immune response to T cell−dependent antigens in Bcl10-deficient mice. Wild-type (Bcl10+/+) and Bcl10-deficient (Bcl10-/-) mice were immunized intraperitoneally with the T cell−dependent antigen, TNP-KLH, and were given a 'booster' immunization 7 d later. Sera were collected 14 d after the initial immunizations, and TNP-specific immunoglobulin isotypes were determined by ELISA. Data are from three mice per genotype. (d) Inability of Bcl10-deficient mice to clear bacteria from their bloodstream. Wild-type (Bcl10+/+; n = 6) and Bcl10-deficient (Bcl10-/-; n = 5) mice were infected intranasally with S. pneumoniae. After this infection, blood samples were collected daily from tail veins for 14 d. Bacterial titers were determined by serial dilution of the blood and subsequent plating on blood agar plates. Average bacterial titers of S. pneumoniae in the blood of wild-type or Bcl10-deficient mice were plotted against time. (e) High mortality of Bcl10-deficient mice after S. pneumoniae infection. The survival percentages of the S. pneumoniae−infected wild-type (Bcl10+/+) and Bcl10-deficient (Bcl10-/-) mice over time are shown as Kaplan-Meier plots. Data shown in d and e are representative of two independent experiments.
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Defective immune responses in Bcl10-deficient mice
Marginal zone and B1 B cells are important in the initiation of a rapid and effective humoral immune response to T cell−independent antigens, whereas follicular B cells participate later in the T cell−dependent antibody responses8. We examined the involvement of Bcl10 in the humoral immune responses to T cell−independent and T cell−dependent antigenic challenge. We injected TNP-Ficoll or a T cell−dependent antigen, TNP-keyhole limpet hemocyanin (KLH), intraperitoneally into Bcl10-deficient or wild-type mice. We measured serum titers of TNP-specific antibodies 7 or 14 d after the TNP-Ficoll or TNP-KLH immunizations, respectively. Wild-type mice produced TNP-specific antibodies, with IgM as a major isotype in response to TNP-Ficoll (Fig. 7b), and IgG1 as the major isotype in response to TNP-KLH (Fig. 7c). In contrast, Bcl10-deficient mice failed to produce any detectable TNP-specific antibodies in response to either TNP-Ficoll or TNP-KLH. These data demonstrate that Bcl10 is essential in the T cell−independent humoral response, presumably because of defects in both marginal zone and B1 B cell function, and in the follicular B cell−initiated T cell−dependent humoral response as well.
We examined the involvement of Bcl10 in the ability of mice to contain an invasive bacterial infection. We infected Bcl10-deficient and wild-type mice intranasally with TIGR4, a virulent strain of S. pneumoniae. This is the natural route of acquisition of this pathogen and routinely results in pneumonia and bacteremia. Containment of the infection requires a rapid innate immune response well in advance of antibody-mediated bacterial opsonization. Bcl10-deficient mice showed an inability to efficiently clear bacteria from their bloodstream, whereas wild-type control mice cleared the bacteria from their blood between 2 and 5 d after infection (Fig. 7d). Consistent with the high titers of bacteria in their bloodstream, all of the Bcl10-deficient mice died within 12 d of infection, whereas more than 80% of wild-type mice survived (Fig. 7e). Half of the deaths occurred before day 6, indicating a defective immune response at a time when antibody was not yet at high titer. These data demonstrate that normal Bcl10 function is crucial for the immune defense against bacterial infection in terms of both antibody-mediated events, which occur 7 d and later after challenge, and early responses during the course of infection.
Discussion
In this study, we have demonstrated that Bcl10 is essential not only for the normal function but also for the maturation of all mature B cell subsets: follicular, marginal zone and B1. The B cell deficiencies we found seemed to be due at least in part to abnormalities in the ability of these cells to activate NF-B-mediated responses. The family of NF-B proteins, which consists of five members, NF-B1 (p50), NF-B2 (p52), c-Rel, RelA (p65) and RelB, is involved in both the development and function of follicular, marginal zone and B1 B cells27. Lack of NF-B1 (p50) impairs marginal zone B cell development28, whereas absence of both NF-B1 and NF-B2 blocks the development of follicular B cells27. In addition, the development of follicular B cells deficient in both c-Rel and RelA is severely impaired29. Therefore, the failure of normal activation of NF-B proteins after B cell receptor ligation in Bcl10-deficient follicular B cells23 and after stimulation with LPS in Bcl10-deficient marginal zone B cells, as we report here, probably contributes to the defective maturation of these subsets of B cells. A previous study reported that Bcl10-deficient splenic B cells proliferate normally in response to LPS stimulation23. Our data, however, showed that only Bcl10-deficient follicular B cells (the wild-type counterparts of which constitute most of the total splenic B cells normally), but not Bcl10-deficient marginal zone B cells, responded normally to LPS. Thus, in addition to its involvement in B cell receptor−mediated signaling23, Bcl10 seems to act in a unique pathway specific for LPS signaling in marginal zone B cells; this pathway seems to be distinct from the signaling mechanism used by LPS in follicular B cells, which seems to be Bcl10-independent.
Other signals (for example, those derived from chemokines30) also participate in the maturation of peripheral B cells. Pyk2, a tyrosine kinase, and Lsc, a Rho GTP exchange factor, are potentially involved in chemokine signaling, and mice lacking Pyk2 (ref. 31) or Lsc32 suffer severe impairment of marginal zone B cell development. In part, the B cell abnormalities found in mice such as those lacking Pyk2 occur because of abnormal motility of these cells in response to chemokines like stromal cell−derived factor 1 that control aspects of B cell migration and compartmentalization31. Similar abnormalities do not seem to contribute to the B cell defects seen in Bcl10-/- mice, however, given that migration assays31 of Bcl10-deficient follicular and marginal zone B cells showed their motility in response to stromal cell−derived factor 1 to be similar to that of the corresponding wild-type cells (data not shown). Nonetheless, although not formally examined here, it is possible that Bcl10 could participate in the activation of NF-B (or perhaps non-NF-B-associated signaling pathways) by as-yet-undefined chemokines to contribute to B cell maturation. Distortion of the splenic microarchitecture by the lack of RelB, an NF-B family member, in nonlymphoid cells, or Dock2, an activator of Rac, is also able to impair marginal zone B cell development33,
34. It seems unlikely, however, that Bcl10 deficiency could indirectly affect B cell maturation by a similar distortion of the splenic microenvironment, given that immature B cells derived from wild-type mice are able to differentiate normally in Bcl10-deficient mice. Rather, the failure of Bcl10-deficient mice to generate marginal zone B cells results from an intrinsic B cell defect.
Both marginal zone and B1 B cells are early participants in T cell−independent immune responses and give rise to the early wave of plasma cells in these responses8. Functionally, marginal zone B cells are able to interact with a wider variety of potential blood-borne antigens8, although B1 B cells also participate in early immune responses against many bacteria and viruses35. In addition, B1 and marginal zone B cells can take part in the T cell−dependent immune response, although mature follicular B cells are principal effectors of this response36. Mice lacking one or two of the three subsets of mature B cells typically have impaired humoral immune responses. For example, Btk-deficient mice, which are deficient in follicular and B1 B cells but have relatively normal marginal zone B cells, show an impaired response to both T cell−dependent and T cell−independent antigens37,
38. Similarly, Pyk-2-deficient mice, which have normal B1 cells but severely reduced marginal zone B cells, respond poorly to T cell−independent antigens31. In contrast, there is a complete absence of the humoral immune response to both T cell−independent- and T cell−dependent-specific antigens in Bcl10-deficient mice, and these mice are very susceptible to S. pneumoniae challenge. These results are consistent with the fact that Bcl10-deficient mice suffer deficiencies of all three subsets of peripheral mature B cells.
Consistent with a previous study of Bcl10-/- mice23, we found Bcl10 to be dispensable for early B-lymphopoiesis; in contrast to that earlier study23, however, we identified severe abnormalities of all three mature B cell populations due to the absence of Bcl10. The reasons for the discrepancies in these findings are not clear, but they do not seem to be because of differences in the technical design of the knockouts or substantial mouse strain variations. The Bcl10-targeting strategies used both here and in the previous study23 produced mice that lack any detectable Bcl10 protein; thus, both deletions produce completely functional null strains. Likewise, both studies used E14 ES cells (129/Ola) to produce chimeric mice, which were then backcrossed to strain C57BL/6 to obtain heterozygous mice that were subsequently intercrossed to generate homozygous-null offspring23.
Human BCL10 was originally identified by the cloning of the t(1; 14) chromosomal translocation involved in MALT lymphomagenesis10,
11. The involvement of BCL10 in the formation of MALT lymphoma is not yet fully understood. Overexpression studies have suggested that BCL10 may exert pro-apoptotic effects in at least some cell types10,
11,
39, and mutations of the gene that result in the synthesis of truncated BCL10 proteins have been reported in t(1; 14)-positive MALT tumors10,
11. Therefore, it has been proposed that inactivation of BCL10 is an underlying mechanism of MALT lymphoma causation. However, our analyses showed that Bcl10 absence did not substantially affect B or T cell apoptosis or cause tumor formation (data not shown). In addition, our in vivo BrdU-labeling experiments showed that Bcl10-deficient follicular and marginal zone B cells had much less labeling than did the corresponding wild-type B cells. Furthermore, Bcl10 function was required for LPS-induced marginal zone B cell and antigen receptor−induced follicular B cell proliferation. Our data indicate that Bcl10 functions as a regulator of the production, rather than the survival, of both follicular and marginal zone B lymphocytes, the latter being the cells from which MALT lymphomas are derived40. Thus, it is likely that up-regulation of normal BCL10 constitutively by the t(1; 14) activates proliferative signals (for example, mediated by NF-B activation and perhaps other signaling pathways), thereby promoting MALT lymphoma development and progression. In support of this hypothesis, overexpression of the normal Bcl10 protein (but not truncated, mutant forms such as those described in some t(1; 14)-positive MALT lymphomas10,
11) in the B cells of transgenic mice led to marginal zone B cell expansion (L.X., X.C. and S.W.M.; unpublished data). Such observations indicate that inhibition of BCL10 function might have therapeutic benefit against MALT lymphomas.
Methods
Generation of Bcl10-/- mice.
An EcoRI fragment of the Bcl10 genomic DNA locus was used to develop the Bcl10 targeting vector, in which the third exon of Bcl10, which encodes almost all of the entire single CARD domain present in the protein, and its flanking sequences were replaced by a neo expression cassette. An EcoRI site was introduced into the targeted locus by the neo cassette. A herpes simplex virus−thymidine kinase gene cassette mediating negative ES cell selection with ganciclovir was inserted in the 5' end of the Bcl10-neo construct. Electroporation of the linearized Bcl10 targeting construct into E14 ES cells was done as described41. Correctly targeted ES cell clones with normal karyotypes were injected into blastocysts to generate chimeric mice for subsequent breeding to obtain germline transmission. Mice were genotyped by Southern blot analysis or PCR. The status of Bcl10 expression in tissues from Bcl10-/- and wild-type mice was assessed by immunoblot with a C-terminal-specific Bcl10 monoclonal antibody (sc-5273; Santa Cruz Biotechnology). Mice derived from two independent targeted ES cell clones were used in the subsequent studies.
Flow cytometry.
Spleen and bone marrow cells obtained from wild-type or mutant mice were made into single-cell suspensions in PBS supplemented with 1% BSA. The cells were then stained with a combination of fluorescence-conjugated antibodies. FITC-conjugated (553818) and purified (553817) rat anti-mouse CD21; PE-conjugated (553139) and biotin-conjugated (553137) anti-CD23; PE-conjugated anti-CD5 (553023); Cy-chrome conjugated anti-CD19 (551001); PE-conjugated anti-rat Ig (550767); and Cy-chrome-conjugated anti-B220 (553091) were all purchased from Pharmingen; all are monoclonal except PE-conjugated anti-rat Ig. Both FITC- and PE-conjugated anti-IgM were purchased from Southern Biotechnology (both monoclonal; 1140-02 and 1140-09, respectively). Samples were applied to a flow cytometer (FACSCalibur, Becton Dickinson), and data were collected and analyzed using CellQuest software (Becton Dickinson).
Immunofluorescent histological analysis.
Spleen tissue was embedded in optimum cutting temperature compound (Lab-Tek Products) and flash-frozen in liquid nitrogen. Sections 5 m in thickness were fixed in cold acetone, air-dried and incubated for 1 h at room temperature in PBS containing 1% BSA, 10% normal rat serum and 10% normal goat serum. Then, the tissue sections were stained at 4 °C overnight with rhodamine (TRITC)-conjugated goat anti-mouse IgM (polyclonal; 1020-03; Southern Biotechnology) and FITC-conjugated rat anti-mouse metallophilic macrophages (MOMA-1; monoclonal; MCA947F; Serotec). After being stained, sections were washed and mounted with Fluormount G (Southern Biotechnology) and were examined with a Leica TCS NT SP confocal laser-scanning microscope equipped with argon (488 nm) and krypton (568 nm) lasers (Leica Microsystems). Images were recorded with Leica PowerScan Software.
FACS analysis of apoptosis (TUNEL).
Splenocytes were stained with FITC-conjugated anti-CD21 and biotin-conjugated anti-CD23 followed by Cy-chrome-labeled streptavidin. Cells were then fixed, permeabilized and labeled with dUTP-Texas red according to the manufacturer's instructions (In Situ Cell Death Detection Kit; Boehringer Mannheim). The degree of TUNEL positivity in the gated B cell subpopulations, follicular (CD21+CD23+) and marginal zone (CD21hiCD23lo), was analyzed by FACS. B cells labeled with dUTP in the absence of terminal transferase served as the negative control. TUNEL-positive cells were defined as those showing fluorescence greater than that of the negative controls.
In vivo BrdU staining assay.
The in vivo BrdU staining assay was done as described42. Mice 8−10 weeks old were injected intraperitoneally with 0.6 mg BrdU (Sigma-Aldrich) in 0.2 ml PBS at 12-hour intervals for 4 d. The splenocytes from BrdU-treated mice were stained with purified rat anti-mouse CD21 followed by PE-conjugated anti-rat immunoglobulin and biotin-conjugated anti-CD23, and finally by Cy-Chrome-labeled streptavidin. Finally, the cells were stained with FITC-conjugated anti-BrdU (monoclonal; 347583; Becton Dickinson). The degree of BrdU positivity in the gated B cell subpopulations, follicular (CD21+CD23+) and marginal zone (CD21hiCD23lo), was analyzed by FACS.
Proliferation assays.
Single-cell suspensions of splenocytes were stained with anti-B220, anti-CD21 and anti-CD23. In the B220+ gate, cells were sorted into CD21+CD23+ (follicular) and CD21+CD23- (marginal zone) B cell populations. The sorted cells (2 105) were stimulated for 24 h with LPS (10 g/ml) in a 96-well plate, and subsequently pulsed for 16 h with [3H]thymidine (1 Ci/well). Samples were collected using a MACH III harvester (TOMTEC), and the incorporation of [3H]thymidine was determined with a Wallac MicroBeta TriLux liquid scintillation system (PerkinElmer).
NF-B gel-mobility-shift assay.
Based on the expression of B220, CD21 and CD23, follicular (CD21+CD23+) and marginal zone (CD21+CD23-) B cells were purified from splenocytes by FACS. After being stimulated with LPS (10 g/ml) for 16 h or anti-IgM (10 mg/ml; polyclonal; 115-005-003; Jackson ImmunoResearch Laboratories) for 24 h, nuclear extracts were prepared from the purified B cells (1 106) and NF-B gel mobility shift assays were done as described43. Nuclear extract (1 g) was incubated for 20 min at room temperature with 32P-labeled NF-B probe (5'-AGTTGAGGGGACTTTCCCAGGC-3'). The band shifts were resolved by 4% polyacrylamide gel electrophoresis at 4 °C.
Bone marrow transplantation.
Bone marrows were isolated from both hind limbs of wild-type or mutant mice and were injected intravenously into sublethally irradiated recipient RAG-1-deficient (Rag1-/-) mice (600 rad) or Bcl10-/- mutant mice (750 rad). Then, 8 weeks later, splenic B cell maturation in the recipient transplanted mice was examined.
Immunizations and serum titer analysis.
Immunization and serum titer analysis were done as described32. Wild-type and Bcl10-deficient mice 11−14 weeks old were immunized intraperitoneally with 100 g T cell−independent antigen (TNP-Ficoll) or 100 g T cell−dependent antigen (TNP-KLH). Serum was collected from mice immunized with TNP-Ficoll 7 d after immunization. Mice immunized with TNP-KLH were given a 'booster' immunization on day 7 and serum was collected 14 d after the initial immunization. TNP-specific antibodies were determined by ELISA and data were analyzed using SigmaPlot software (SPSS Science).
Bacterial infections.
S. pneumoniae, serotype 4, strain TIGR4 (ref. 44) was cultured overnight at 37 °C in C+Y (casein hydrolysate plus yeast extract) media45 in an atmosphere of 5% CO2. The following morning, 100 l were transferred to 10 ml fresh C+Y media and samples were incubated at room temperature until the culture reached an optical density of 0.5 at a wavelength of 620 nm. Pneumococci were pelleted and washed with sterile PBS (BioWhittaker). Bacteria were suspended in PBS at a concentration corresponding to 1 109 colony-forming units/ml, and the concentration of bacteria was confirmed by serial dilution of the suspension and subsequent growth on tryptic soy agar plates (Difco Laboratories) supplemented with 3% defibrinated sheep blood (Micropure Medical). Mice were anesthetized by subcutaneous injection of MKX, consisting of 1 ml ketamine (100 mg/ml; Fort Dodge Laboratories), 5 ml xylazine (100 mg/ml; Miles Laboratories) and 21 mL PBS, at a dose of 0.05 ml per 10 gm body weight. Once anesthetized, mice were positioned upright and challenged intranasally with 25 l of the bacterial suspension. Infected mice were monitored for 14 d and blood samples were collected daily from the tail vein. Bacterial titers were determined by serial dilution of the blood and subsequent plating on blood agar plates.
Received 17 March 2003; Accepted 10 July 2003; Published online: 10 August 2003.
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